Introduction

In industrialised countries of Europe and the United States, Salmonella has been the bacteria most frequently associated with human diarrhoea. In the last years the epidemiology of several infectious disease has changed dramatically, making necessary the continuous monitoring of some pathogens, such as Salmonella. To help assessing the burden of Salmonella infection over the last years in Gipuzkoa and to help public pealth officials detect possible causes of these changes, the incidence of human enteric infection involving Salmonella serovars was studied in a stable population over an 18-year period.

Methods

Population data

Gipuzkoa is a province located in northern Spain (Basque Country), bordered by the Bay of Biscay and France to the north. Gipuzkoa was divided into seven different health care-administrative districts. The study population was that of the city of San Sebastián (capital of the province) and three other neighbouring districts. This represented about half of the entire population of Gipuzkoa (675 529 people in 1991) and was nearly stable, ranging between 361 861 and 355 515 inhabitants. The changes in population were primarily due to births and deaths, as no significant migratory movements were observed. This population data was obtained from the 1986, 1991 and 1996 official population records of the Basque Institute of Statistics.
No significant changes in medical assistance or diagnostic procedures were introduced during the study period. All stool culture samples were processed in the same Microbiology Department. This laboratory operates under the National Insurance System, which nearly covers 100% of the population. Other laboratories, including private laboratories, perform analyses of less than 1% of the samples from the paediatric population and between 5-10% of the samples from the adult population.

Case definition

During the study period, all the patients who sought medical care for gastroenteritis and sent one or more stool specimens to the laboratory were recruited for the study. This included only cultured confirmed cases and only the first Salmonella isolate from each patient. Asymptomatic carriers were excluded. A case of Salmonella enteric infection was defined as a patient with a non-Typhi Salmonella stool isolate who sought medical care for gastroenteritis (2411 Salmonella positive stools were rejected during those years as they were repeated positive cultures of an already diagnosed patient). All patients included were assigned to two major groups: 'paediatric' (=14 years old) and 'adult' (>14 years old). The paediatric population was further subdivided into two age groups: <2 years and 2-14 years.
Calculation of annual incidence rate
In this study, annual incidence rate was defined as the annual number of patients suffering from gastrointestinal illness with a Salmonella stool isolate, divided by the population for that year, and stated as the rate per 100 000 inhabitants/year. The population used was that of the nearest year in which official data was available. Therefore, these are minimum incidence values, as not all cases of Salmonella enteric disease which occurred in the population studied were sent for stool cultures.
Microbiological procedures
Stool cultures were performed using standard selective and enrichment culture techniques. All Salmonella-like colonies were identified to the genus level by their biochemical characteristics using the API 20 E® (BioMérieux, France) system. Salmonella serovars were established by slide agglutination using both polyvalent and specific rabbit sera (Pasteur Diagnostics, France) against somatic (O) and flagellar (H) antigens according to the Kauffmann-White scheme.
Statistical methods
Statistical analysis was conducted with a Stata software (release 6, Stata Corporation). The confidence intervals for incidence rates were calculated assuming that the number of cases was compatible with a Poisson probability distribution law. The relative risk (RR) was estimated based on a Poisson regression model with only two independent variables: age of the patient and calendar year of isolation. Therefore, the RR estimated for a specific calendar year is adjusted by the effect of age on incidence. The reference age group was the adult population, and 1983 the reference year.
Results
From January 1983 to December 2000, enteric disease due to Salmonella was detected in 7734 patients (table 1). The mean annual rate of cultured-confirmed salmonellosis in the overall population during the 18-year study was 120.17 cases per 100 000 (CI 95%; 117.50-122.85). Figure 1 shows the annual rate of all Salmonella serovars, Salmonella Enteritidis and Salmonella Typhimurium over years. The exact age was known in 6911 patients (89.4%). After proportionally distributing the 224 paediatric patients with unknown ages between the two paediatric age groups defined, the mean annual rate of Salmonella enteric infection was 1120.72 cases (CI 95%; 1060.42-1181.03) per 100 000 in children under the age of 2 years old, 255.96 cases (CI 95%; 246.44-265.49) per 100 000 for the population aged between 2 and 14 years, and 69.45 cases (CI 95%; 67.19-71.70) per 100 000 for those aged over 14.
The annual rate of Salmonella enteric infection distributed by age group is showed in table 2. These rates decreased to the half between 1990 and 1994 for the whole population, reflecting basically what happened with the group of adult population. Over the period studied, trends of the relative risk for Salmonella infections followed an undulating pattern for all Salmonella serovars and for Salmonella Enteritidis (figure 2). The year with the highest overall relative risk of Salmonella and of Salmonella Enteritidis infections was 1990. Between 1983 and 1990, the relative risk of S. Enteritidis infections increased 4.7-fold. From 1990 to 1994, it gradually decreased, and re-increased since then.
Statistical differences in the relative risk of Salmonella infection were observed between the different age groups. The relative risk of the population aged 2-14 years old and under 2 years old versus the population over 14 years old was 3.7 (CI 95%; 3.52-3.89) and 16.2 (CI 95%; 15.2-17.3) respectively (p<0.0001). Two Salmonella enterica serovars, Enteritidis and Typhimurium, accounted for 92.1% (6219 and 907 cases respectively) of all salmonellosis, followed in order of frequency by S. Infantis (169 cases, 2.19%) and S. Hadar (68 cases, 0.88%).

Table 1
Distribution by age group of number of patients with Salmonella enteric infection

Table 2
Rate per 100 000 of Salmonella enteric infections by age group

Population included for 1983-88, 1989-93 and 1994-2000 was respectively 7308, 6104 and 6291 for children < 2 years ;
79 200, 59 896 and 44 094 for children 2-14 years old ; and 275 353, 289 458 and 305 130 for adults (> 14 years old).

Discussion

Most studies on the incidence of Salmonella infections undertaken since the early 1980's have shown that S. Enteritidis and S. Typhimurium are the most frequently isolated serovars in Europe and in the United States (1,2). In previous decades, the prevalence of Salmonella Enteritidis in Europe was low, and the incidence of this infection throughout Europe during the last two decades was associated with the spreading of this infection among poultry (2). In Gipuzkoa the incidence of S. Typhimurium was higher than that of S. Enteritidis in the late 1970's and the first two years of the 1980's (data not shown). However, as shown in this study, since 1984 the annual rate of S. Enteritidis infections was more than 3 times greater than the rate of S. Typhimurium. The noteworthy increase in the rate of enteric infections associated with S. Enteritidis during the first years of this study is similar to that observed during the first half of the 1980´s in other regions of Spain (3), and in other European countries (2,4).
In our study, as of 1983 the overall rate of Salmonella infections increased due to the increase in the incidence of just one serovar, Salmonella Enteritidis, which was more frequently isolated each year than all the others, and which was mostly involved in the undulating trends observed overall. Between 1991-94 the infection trend decreased, and then increased again during the latter years of the study. Although there are many factors that may have contributed to the decrease observed between 1991 and 1994, we consider that it may be related in part to an Order issued by the Health Department in 1991 regarding the preparation and conservation of products for public consumption that contain raw egg, such as mayonnaise. This Order, which was compulsory for restaurants and other public facilities, was highly publicised by the media and had a significant effect among the overall population. Unfortunately, the educational effect of the debate in the media regarding this and other preventive measures referred to food handling practices was probably lost over time.
In the last decade, a European surveillance observed a declining trend in the incidence of Salmonella Enteritidis infections in Western Europe between 1993 and 1995 (5,6), followed by an increase from the second half of 1996 into early 1998. This trend reversed over the remaining months. However, this latter decrease did not occur in Spain, which was one of the four countries where an increase was recorded (7).
In the Gipuzkoa province, the mean annual rate of Salmonella infection in the two paediatric age groups (1121 children under 2 years-old, and 256 children from 2-14 years old) was more than 10-fold that reported from 1987-1997 in the United States (8). Salmonella Typhimurium was the most frequent serovar in the United States. The rates of Salmonella Typhimurium infection in Gipuzkoa were much lower than those of Salmonella Enteritidis, but 4-5 times higher than those reported in the United States (1,8). The USA data as well as ours are limited to laboratory-confirmed illnesses, but our laboratory-based surveillance system was passive whereas the American system was active.
The high rates of Salmonella enteric infections observed in our area are of great concern. Further improvements in the microbiological quality of food, especially during the early stages of the food chain, as well as more active educational programs for food handling and preparation by professional and home cooks, need to be implemented by the Public Health authorities in order to reduce the risk of human Salmonella infection in our region.